[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 28 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

November 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-(aminocarbonyl)-1-ß-D-ribofuranosyl-pyridinium bromide

Cas Number:

78687-39-5

Molecular formula:

C11H15BrN2O5

IUPAC Name:

3-(aminocarbonyl)-1-ß-D-ribofuranosyl-pyridinium bromide

impurity 1

Reference substance name:

Nicotinamide

EC Number:

202-713-4

EC Name:

Nicotinamide

Cas Number:

98-92-0

Molecular formula:

C6H6N2O

IUPAC Name:

nicotinamide

impurity 2

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

impurity 3

Reference substance name:

Ethyl acetate

EC Number:

205-500-4

EC Name:

Ethyl acetate

Cas Number:

141-78-6

Molecular formula:

C4H8O2

IUPAC Name:

ethyl acetate

impurity 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 5

Reference substance name:

inorganic salts by sulphated ash

IUPAC Name:

inorganic salts by sulphated ash

impurity 6

Reference substance name:

Bromic acid

EC Number:

232-158-3

EC Name:

Bromic acid

Cas Number:

7789-31-3

Molecular formula:

BrHO3

IUPAC Name:

bromic acid

impurity 7

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

Test material form:

solid

Details on test material:

Batch / Lot No. 1000146/01

Specific details on test material used for the study:

Batch-No.: 1000146/01
Storage: Freezer

Method

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (Escheria coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

bacteria

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

bacteria

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) rat liver S9 mix
The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

Test concentrations with justification for top dose:

Initial Mutation Test and Confirmatory Mutation Test (±S9): 5000, 1600, 500, 160, 50 and 16 µg/plate

Concentration Range Finding Test (Informatory Toxicity Tests):
Based on the solubility test, the stock solution with a concentration of 50 mg/mL was prepared in ultrapure water and diluted in at least 6 steps by factor of approximately 10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
The revertant colony numbers of vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the performed Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case; all of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9).

Vehicle / solvent:

- Vehicle/solvent used: Ultrapure water
- Justification for choice of solvent/vehicle: In the solubility test the test item behaviour was investigated in the applied test system. The test item was adequately soluble in ultrapure water (ASTM Type I); therefore, it was dissolved in ultrapure water, accordingly. The obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

Positive control concentration: 4 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control concentration: 2 µg/plate for TA100 and TA1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control concentration: 50 µg/plate for TA1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control concentration: 2 µg/plate for WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

Positive control with metabolic activation concentration: 2 µg/plate for S. typhimurium strains; 50 µg/plate for E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Initial mutation test: in agar (plate incorporation)
- Confirmatory test: in agar (plte incorporation) with preincubation
- Bacterial Cultures: The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 11-12 hours in a 37°C Benchtop Incubator Shaker.
- Molten top agar preparation: Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).
The content of the tubes:
top agar 2000 µL
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain* 100 µL
phosphate buffer (pH: 7.4) or S9 mix 500 µL
*: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA.
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls.
- Exposure duration: The plates were incubated at 37°C for about 48 hours.
-Preincubation period (in confirmatory test): Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to allow direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, two mL of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates.

NUMBER OF REPLICATES
Triplicates

METABOLIC ACTIVATION SYSTEM
The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

VALIDITY CRITERIA
The tests (initial and confirmatory mutation tests) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 10^9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The positive control reference items (known mutagens) show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

- For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate. For test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest dose tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment.
Insolubility is assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.
- The test has to include five analyzable concentrations (where the precipitate does not interfere with the scoring) and a minimum of three non-precipitated dose levels.

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Statistics:

According to the guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Validity criteria: All criteria for the validity of the performed experiments were met.

- Controls: In the performed main experiments multiple test items were tested with reference values from the common parallel controls.
The spontaneous revertant colony numbers of the ultrapure water (ASTM Type I) vehicle control plates showed characteristic mean numbers in line with the actual historical control data ranges in all strains in both main experimental phases of the study.
Each of the investigated reference mutagens (positive controls) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in main experimental phases and additionally the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in main experimental phases, in all tester strains.
The revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in the main experiments were slightly higher or lower than the ultrapure water (ASTM Type I) control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.

-Initial and Confirmatory Mutation Tests (Plate incorportation and pre-incubation Tests): No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Nicotinamide-beta-D-riboside bromide at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. In the main experiments, occasional increases in the revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest mean revertant colony number increase was observed in the confirmatory mutation test (pre-incubation test) in Salmonella typhimurium TA1535 strain at 16 µg/plate, in the absence of exogenous metabolic activation (-S9). The revertant colony numbers at this concentration remained within the corresponding vehicle historical control data range; the value was unique, was not accompanied with dose-relationship; therefore, this higher value was considered rather as being within the biological variability range of the applied system than a test item effect. The mutation rate was 1.63, far below the genotoxicological threshold for being positive.

In the performed initial and confirmatory mutation tests, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Nicotinamide-beta-D-riboside bromide (CAS Nr. 78687-39-5) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

Purpose of the study:

The test item Nicotinamide-beta-D-riboside bromide (CAS Nr. 78687-39-5) was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

Bacterial strains, exogenous metabolic activation:

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/ß-naphthoflavone-induced rats.

Experimental phases:

The study included preliminary solubility investigations, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Vehicle, test item concentrations, rationale for dose selection:

Based on the results of the solubility test and the concentration range finding test, the test item was dissolved in ultrapure water (ASTM Type I) and the following concentrations were prepared and investigated in the initial and confirmatory mutation tests in the absence and presence of exogenous metabolic activation:

±S9: 5000; 1600; 500; 160; 50 and 16 µg/plate.

The selection of the concentration range was in accordance with the OECD 471 guideline. The maximum test concentration was in all strains 5000 µg/plate, as recommended in the guideline for soluble non-toxic compounds.

At the preparation of the test item stock solutions any correction (multiplier) factor was not taken into consideration, the doses were based on the final formulation as is.

Solubility, precipitation:

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.

Cytotoxicity results:

In the performed experiments inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.

Mutagenicity results:

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Nicotinamide-beta-D-riboside bromide at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Conclusion:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

In conclusion, the test item Nicotinamide-beta-D-riboside bromide (CAS Nr. 78687-39-5) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.